JOURNAL OF BACTERIOLOGY, Oct. 2010, p.

50025017
0021-9193/10/$12.00 doi:10.1128/JB.00542-10
Copyright 2010, American Society for Microbiology. All Rights Reserved.

Vol. 192, No. 19

The Human Oral Microbiome

Floyd E. Dewhirst,1,2* Tuste Chen,1 Jacques Izard,1,2 Bruce J. Paster,1,2 Anne C. R. Tanner,1,2
Wen-Han Yu,1 Abirami Lakshmanan,1 and William G. Wade1,3
Department of Molecular Genetics, The Forsyth Institute, Cambridge, Massachusetts 021421; Department of Oral Medicine,
Infection and Immunity, Harvard School of Dental Medicine, Boston, Massachusetts 021152; and Kings College London
Dental Institute at Guys, Kings College and St. Thomas Hospitals, Infection Research Group, Guys Campus,
London SE1 9RT, United Kingdom3
Received 11 May 2010/Accepted 10 July 2010

from within the oral cavity. Studies have shown that different
oral structures and tissues are colonized by distinct microbial
communities (2, 39). Approximately 280 bacterial species from
the oral cavity have been isolated in culture and formally
named. It has been estimated that less than half of the bacterial
species present in the oral cavity can be cultivated using anaerobic microbiological methods and that there are likely 500
to 700 common oral species (47). Cultivation-independent molecular methods, primarily using 16S rRNA gene-based cloning
studies, have validated these estimates by identifying approximately 600 species or phylotypes (47; http://www.homd.org).
The oral cavity is a major gateway to the human body. Food
enters the mouth and is chewed and mixed with saliva on its
way to the stomach and intestinal tract. Air passes through the
nose and mouth on the way to the trachea and lungs. Microorganisms colonizing one area of the oral cavity have a significant probability of spreading on contiguous epithelial surfaces
to neighboring sites. Microorganisms from the oral cavity have
been shown to cause a number of oral infectious diseases,
including caries (tooth decay), periodontitis (gum disease),
endodontic (root canal) infections, alveolar osteitis (dry
socket), and tonsillitis. Evidence is accumulating which links
oral bacteria to a number of systemic diseases (58), including
cardiovascular disease (6, 32), stroke (31), preterm birth (46),
diabetes (25), and pneumonia (4).
For most of the history of infectious diseases, medical practitioners focused on individual organisms in pure culture
through the perspective of Kochs postulates. With the realization that essentially all surfaces of humans, animals, plants, and
inanimate objects, which have air or water interfaces, are cov-

The microorganisms found in the human oral cavity have

been referred to as the oral microflora, oral microbiota, or
more recently as the oral microbiome. The term microbiome
was coined by Joshua Lederberg to signify the ecological
community of commensal, symbiotic, and pathogenic microorganisms that literally share our body space and have been all
but ignored as determinants of health and disease (37). The
term microbiome has been embraced by the Human Microbiome Project and investigators who believe an understanding of
human health and disease is impossible without fully understanding the collective microbiome/human superorganism.
This work describes the identification and phylogeny of the
most prevalent oral taxa.
The oral cavity, or mouth, includes several distinct microbial
habitats, such as teeth, gingival sulcus, attached gingiva,
tongue, cheek, lip, hard palate, and soft palate. Contiguous
with the oral cavity are the tonsils, pharynx, esophagus, Eustachian tube, middle ear, trachea, lungs, nasal passages, and
sinuses. We define the human oral microbiome as all the microorganisms that are found on or in the human oral cavity and
its contiguous extensions (stopping at the distal esophagus),
though most of our studies and samples have been obtained

* Corresponding author. Mailing address: The Forsyth Institute, 245

First St., Cambridge, MA 02142. Phone: (617) 892-8298. Fax: (617)
892-8432. E-mail: fdewhirst@forsyth.org.
Supplemental material for this article may be found at http://jb
.asm.org/.

Published ahead of print on 23 July 2010.

The authors have paid a fee to allow immediate free access to
this article.
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The human oral cavity contains a number of different habitats, including the teeth, gingival sulcus, tongue,
cheeks, hard and soft palates, and tonsils, which are colonized by bacteria. The oral microbiome is comprised
of over 600 prevalent taxa at the species level, with distinct subsets predominating at different habitats. The
oral microbiome has been extensively characterized by cultivation and culture-independent molecular methods
such as 16S rRNA cloning. Unfortunately, the vast majority of unnamed oral taxa are referenced by clone
numbers or 16S rRNA GenBank accession numbers, often without taxonomic anchors. The first aim of this
research was to collect 16S rRNA gene sequences into a curated phylogeny-based database, the Human Oral
Microbiome Database (HOMD), and make it web accessible (www.homd.org). The HOMD includes 619 taxa
in 13 phyla, as follows: Actinobacteria, Bacteroidetes, Chlamydiae, Chloroflexi, Euryarchaeota, Firmicutes, Fusobacteria, Proteobacteria, Spirochaetes, SR1, Synergistetes, Tenericutes, and TM7. The second aim was to analyze
36,043 16S rRNA gene clones isolated from studies of the oral microbiota to determine the relative abundance
of taxa and identify novel candidate taxa. The analysis identified 1,179 taxa, of which 24% were named, 8% were
cultivated but unnamed, and 68% were uncultivated phylotypes. Upon validation, 434 novel, nonsingleton taxa
will be added to the HOMD. The number of taxa needed to account for 90%, 95%, or 99% of the clones examined
is 259, 413, and 875, respectively. The HOMD is the first curated description of a human-associated microbiome and provides tools for use in understanding the role of the microbiome in health and disease.

VOL. 192, 2010

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MATERIALS AND METHODS

This report is based on analysis of 16S rRNA gene sequences from 36,043
clones and over 1,000 isolates. Both clone-based and culture-based studies were
performed under appropriate Institutional Review Board approval. The 16S
rRNA gene sequences, from both clone- and culture-based studies, were obtained in our laboratories over the past 20 years. The protocols listed below
represent our current methods.
Bacterial culture studies. The bacterial isolates came from studies targeting a
wide range of oral health and disease statuses, including periodontitis, caries,
endodontic infections, and noma. The strains were provided mostly by the laboratories of Tanner, Socransky, and Wade and from the culture collection of
Lillian V. Holdeman Moore and the late W. E. C. (Ed) Moore.
Fresh isolates or strains were cultured on BHIHK (brain heart infusion agar
[Becton, Dickinson and Co., Sparks, MD] at 26 g, yeast extract at 5 g, and hemin
at 2.5 mg, and menadione at 250 g in 500 ml H2O plus 25 ml sheeps blood
[Northeast Laboratory Services, Winslow, ME]), FAA (fastidious anaerobe agar
[Acumedia Manufacturers, Inc. Lansing, MI] at 26 g, yeast extract at 5 g, and
hemin at 2.5 mg in 500 ml H2O plus 25 ml sheeps blood), and BUA (Biolog
universal agar [Biolog Inc., Hayward, CA] at 26 g in 500 ml H2O supplemented
with 25 ml sheeps blood). Subsequent passages were performed using the medium on which the strain grew best. Strain identification was performed by the
Touch PCR method, where a wire probe or pipette tip was just touched to the
colony and a minute sample was transferred directly to a tube for amplification
of the 16S rRNA gene operons (see PCR details below). If this quick method did
not work, a loopful of cells was collected and placed in 50 l of 50 mM Tris
buffer, pH 7.6, with 1 mM EDTA and 0.5% Tween 20. Proteinase K (200 g/ml)

was added and incubated at 55C for 2 h. Proteinase K was inactivated by being
heated at 95C for 5 min. A total of 1 l of this preparation was used for PCR.
16S rRNA gene clone library-based studies. Table S1 in the supplemental
material describes the study source for all clones. Specific details on patient
populations, sampling protocols, and sequencing methods used in published
studies are given in the references listed in this table. In brief, 16S rRNA gene
clone libraries were created from and analyzed in unpublished studies and the
following published studies: treponemes in a subject with severe destructive
periodontitis (10); treponemes from several subjects with periodontitis and acute
necrotizing ulcerative gingivitis (ANUG) (18); subgingival plaque from healthy
subjects and subjects with periodontitis, HIV periodontitis, and acute necrotizing
ulcerative gingivitis (ANUG) (47); dental plaque from children with caries (7);
endodontic lesions (45); subjects with advanced noma lesions (49); subjects with
necrotizing ulcerative periodontitis in HIV-positive subjects (1, 50); dorsum
tongue microbiota in subjects with halitosis (36); dental caries in adults (44);
normal biota of healthy subjects at subgingival, supragingival, dorsal tongue,
ventral tongue, hard palate, vestibule, and tonsil sites (2); periodontitis in adults
(15); aggressive periodontitis (22); caries-active and caries-free twins (14); root
caries in elderly subjects (52); ventilator-associated pneumonia (5).
DNA purification from clinical samples. Dental plaque from teeth or subgingival periodontal pockets was collected using sterile Gracey curettes. Plaque
from the curette was transferred into 100 l of TE buffer (50 mM Tris-HCl, pH
7.6; 1 mM EDTA). Bacteria on soft tissues were sampled using nylon swabs. The
material from the swab was dispersed into 150 l of TE buffer. DNA extraction
was performed using the UltraClean microbial DNA isolation kit (Mo Bio
Laboratories, Carlsbad, CA) by following the manufacturers instructions for the
isolation of genomic DNA from Gram-positive bacteria.
16S rRNA gene amplification. Purified DNA samples were generally amplified
with universal primers F24/Y36 to construct broad-coverage libraries. The sequences of primers are given in Table S2 in the supplemental material. Additional libraries seeking expanded coverage of Bacteroidetes/TM7/SR1 groups or
Spirochaetes/Synergistetes groups were amplified with F24/F01 or F24/M98 selective primers, respectively. PCR was performed in thin-walled tubes using a
PerkinElmer 9700 Thermo Cycler. The reaction mixture (50 l, final volume)
contained 1 l of the purified DNA template, 20 pmol of each primer, 40 nmol
of deoxynucleoside triphosphates (dNTPs), 2.5 unit of Platinum Taq polymerase
(Invitrogen, Carlsbad, CA), and 5 l 10 PCR buffer (200 mM Tris-HCl, pH 8.4;
500 mM KCl). A hot-start protocol was used in which samples were preheated at
94C for 4 min, followed by amplification using the following conditions: denaturation at 94C for 45 s, annealing at 60C for 45 s, and elongation at 72C for
2 min, with an additional 1 s for each cycle. Thirty cycles were performed,
followed by a final elongation step at 72C for 15 min. Amplicon size and amount
were examined by electrophoresis in a 1% agarose gel stained with SYBR Safe
DNA gel stain (Invitrogen, Carlsbad, CA) and visualized under UV light. After
verification that a strong amplicon of the correct size was produced, a preparative
gel was run, and the full-length amplicon band was cut out and DNA purified
using a Qiagen gel extraction kit (Qiagen, Valencia, CA).
Cloning procedures. Size-purified 16S rRNA gene amplicons were cloned
using a TOPO TA cloning kit (Invitrogen, Carlsbad, CA) by following the
manufacturers instructions. Transformation was performed using competent
Escherichia coli TOP10 cells provided by the manufacturer. Transformed cells
were plated onto Luria-Bertani agar plates supplemented with kanamycin (50
g/ml) and incubated overnight at 37C.
Library screening. Approximately 90 colonies were picked for each library and
were placed into tubes containing 40 l of 10 mM Tris-HCl, pH 8.0. A total of
1 l of the cell suspension was used directly as the template for PCR with
Invitrogen vector M13 (21) forward and M13 reverse primers. Electrophoresis
on a 1% agarose gel was used to verify the correct amplicon size. PCR product
for preliminary sequencing with primer Y31 (positions 519 to 533, reverse) was
treated with exonuclease and shrimp alkaline phosphatase to remove primers
and dNTPs. Five microliters of PCR product was combined with 0.4 l exonuclease I (10 U/l; USB Corporation, Cleveland, OH) and 0.4 l shrimp alkaline
phosphatase (1 U/l; USB Corporation, Cleveland, OH). The reaction mixture
was incubated at 37C for 15 min and then deactivated at 85C for 15 min. The
PCR products from clones chosen for full sequencing with eight additional
primers were further concentrated and purified using QIAquick PCR purification kits (Qiagen, Valencia, CA).
16S rRNA gene sequencing. Purified DNA was sequenced using an ABI Prism
cycle sequencing kit (BigDye Terminator cycle sequencing kit) on an ABI 3100
genetic analyzer (Applied Biosystems, Foster City, CA). The sequencing primers
(see Table S2 in the supplemental material) were used in quarter-dye reactions
by following the manufacturers instructions.

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ered by complex microbial biofilms (26), microbiologists have

refocused on microbial communities (30). Caries, periodontitis, otitis media, and other infections are now recognized to be
caused by consortia of organisms in a biofilm rather than a
single pathogen (30). It is the premise of the NIH-supported
Human Microbiome Project (51, 64) that we need to know the
identity of all of the major organisms comprising the human
microbiome to fully understand human health and disease and
that we must have tools to rapidly identify members of the
microbiome to carry out meaningful clinical research. Cultureindependent approaches, like the 16S rRNA gene-based molecular cloning methods, have largely replaced cultivation studies for this task, as the molecular methods can reveal the
identities of currently uncultivated microorganisms.
While 16S rRNA gene clone studies have revealed the hidden diversity of the microbial world, clone sequences with no
taxonomic anchors currently fill GenBank, and articles refer to
novel phylotypes by reference to cryptic clone numbers (69).
Taxa known only as 16S rRNA phylotypes cannot be formally
named, as naming requires growth and full phenotypic characterization. Candidatus status can be used to name uncultivated or mixed-culture organisms but still requires characterization significantly beyond a 16S rRNA sequence. Thus, there
is a critical need for a provisional naming system for species/
phylotypes of the human microbiome, so that investigators and
the literature can point to provisionally named taxa rather than
clone sequences.
The first goal of this research was to develop a provisional
taxonomic scheme for the unnamed human oral bacterial isolates and phylotypes and provide this information in an online
publicly available database, namely, the Human Oral Microbiome Database (HOMD) (www.homd.org). The second goal
was to analyze the 36,043 16S rRNA gene oral clone sequences
available from our laboratories to determine the number of
clones observed for each human oral taxon and to identify
additional taxa not included in the initial setup of the HOMD.

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ing clone sequence was compared by BLASTN to the reference sequence(s) and,
if matched, added to that taxon folder. If the BLASTN match failed, the clone
sequence was used to establish a new taxon folder and added to the reference
sequence list. The scripts for these analyses can be obtained from T. Chen.
Extended human oral taxon numbers (A01 to H70) were assigned for each novel
cluster/folder.
Nucleotide sequence accession numbers. The 16S rRNA gene sequences for
the 34,753 clones analyzed are available for download at the Human Oral
Microbiome Database website (http://www.homd.org) and from GenBank under
accession numbers GU397556 to GU432434. GenBank accession numbers for
each taxon in the seven phylogenetic tree figures are included with each taxon
label. Additional full-length 16S rRNA gene sequences deposited for this work
include GenBank accession numbers FJ577249 to FJ577261, FJ717335,
FJ717336, FJ7173350, GQ131410 to GQ131418, and GU470887 to GU470911.

RESULTS AND DISCUSSION

Human Oral Microbiome Database. The backbone of the
HOMD is its set of reference 16S rRNA gene sequences, which
are used to define individual human oral taxa and to create
the phylogenetic and taxonomic structures of the database.
The initial reference set of 16S rRNA gene sequences for the
HOMD consisted of over 800 full-length sequences (each of
which is greater than 1,500 bases) of named oral bacteria from
the oral microbiological literature and strain and clone phylotypes generated from our sequencing and cloning studies. After entering and aligning these sequences in our RNA database, neighbor-joining trees were generated. A total of 78
chimeras were identified and removed from the database. Using a 98.5% sequence similarity cutoff for defining a phylotype,
the approximately 800 sequences were placed in 619 taxa. Each
taxon was given a human oral taxon (HOT) number (arbitrary
number starting at 001). All of the human oral taxa were
placed in a full taxonomic classification, including domain,
phylum, class, order, family, genus, and species, which can be
viewed in Table S4 in the supplemental material. Placement of
species in higher taxa was based solely on tree position. For
example, Eubacterium saburreum was placed in the family
Lachnospiraceae and not Eubacteriaceae. Eubacterium saburreum is not closely related to the type strain of the genus
Eubacterium, Eubacterium limosum, and will need to be placed
in a new genus and subsequently renamed. The HOMD taxonomy is one of several selectable taxonomies available at the
Greengenes website (16) (http://greengenes.lbl.gov). When the
HOMD is selected as the taxonomy, the operational taxonomic
unit (OTU) numbers equal the human oral taxon numbers.
A summary of the phylogenetic distribution of these 619 taxa
is presented in Table 1. It is notable that 65.6% of the taxa
have been cultured. This percentage greatly exceeds that in
many natural environments where less than 1% has been cultivated. The six major phyla, Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteria, Spirochaetes, and Fusobacteria, contain 96% of the taxa. The remaining phyla, Euryarchaeota,
Chlamydia, Chloroflexi, SR1, Synergistetes, Tenericutes, and
TM7, contain the remaining 4% of the taxa.
A detailed presentation of the phylogenies of the 619 oral taxa
can be seen in the phylogenetic trees shown in Fig. 1 to 7. The
percentage of times a node was present in the resampling is
shown only when it was greater than 50%. The name of each
taxon is followed by its designated HOT number, clone or
strain number, GenBank accession number, number of clones

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16S rRNA data analysis. Sequence information determined using primer Y31
(positions 519 to 533, reverse) allows preliminary identification of clones. Clones
or strains whose sequences appeared novel (differing by more than 7 bases from
previously identified oral reference sequences in the first 500 bp) were fully
sequenced on both strands (approximately 1,540 bases) using 6 to 8 additional
sequencing primers (see Table S2 in the supplemental material). Sequences were
assembled from the ABI electropherogram files using Sequencher (Gene Codes
Corporation, Ann Arbor, MI).
Aligned 16S rRNA database. All full-length human oral 16S rRNA gene
sequences which we believed represented novel taxa and those of named human
oral species available in GenBank were entered into a new Aligned Reference
Sequence Database. More than 100 nonoral sequences were also entered to link
oral phylogenetic clusters to named taxa. The basic program set for data entry,
editing and sequence alignment, secondary structure comparison, similarity matrix generation, and phylogenetic tree construction was written by F. E. Dewhirst
in Microsoft QuickBasic and has been previously described (48) (the program is
available from F. E. Dewhirst). Trees for this work were made by exporting
aligned sequences from our database into MEGA version 4 (60). Similarity
matrices were corrected for multiple base changes at single positions by the
method of Jukes and Cantor (33). Similarity matrices were constructed from
the aligned sequences by using only those sequence positions for which 95%
of the strains had data. Phylogenetic trees were constructed using the neighbor-joining method of Saitou and Nei (56). Bootstrapping was performed
using 1,000 resamplings.
Creation of the human oral microbiome 16S rRNA gene reference set. The
sequences for named species, isolates, and clones were obtained primarily by
sequencing efforts in our laboratories or from GenBank. The list of named oral
organisms was compiled from the literature and relied heavily on literature
reports from investigators at the Forsyth Institute (20, 21, 59, 61, 62) and from
Lillian Holdeman Moore and W. E. C. Moore (41, 42, 43), formerly at the
Anaerobe Laboratory at the Virginia Polytechnic Institute. To the initial list of
oral microorganisms, we added exogenous pathogens, such as Corynebacterium
diphtheriae, Bordetella pertussis, Treponema pallidum, Neisseria gonorrhoeae, and
several other species which are causative agents of oral lesions and diseases. For
the 16S rRNA gene sequences of strains or clones that did not match the named
species, we created novel 16S rRNA gene-based phylotypes. We define a phylotype as a cluster of full-length 16S rRNA gene sequences that have greater than
98.5% similarity to one another (23 base mismatches per 1,540 bases) and have
less than 98.5% similarity to neighboring taxa (species or phylotypes). Each
species and phylotype was assigned a human oral taxon (HOT) number, starting
at 001. Prior to assigning the HOT numbers, all provisional sequences were
compared, and those with sequences having greater than 98.5% similarity were
merged into single taxa, except for validly named species, which retained individual HOT numbers regardless of rRNA gene sequence similarity. Sequences
were checked for the possibility of being chimeric using multiple methods.
Neighbor-joining trees were generated using the first 600 bases and compared
with trees using the last 900 bases. Taxa that changed position in the two trees
were further examined with the Chimera Check program at the Ribosomal
Database Project (11) and with Mallard (3). The first and last 100 bases of all
clone sequences described below were analyzed by BLASTN analysis against the
HOMD Reference Set. The distance between end matches was captured from a
distance matrix file for all full-length HOMD references sequences. All sequences with ends being more than 10% different were rejected as chimeric. The
script for this program is available from us. The HOMD reference sequences and
clone sequences were rescreened using Chimera Slayer (courtesy of Brian Haas,
the Broad Institute [http://sourceforge.net]).
Analysis of 16S rRNA gene sequences obtained from clone studies. Clone
sequences were subject to BLASTN analysis against the HOMD Reference
Sequence Set (version 10). Because the first 500 bases of the 16S rRNA molecule
generally contain almost half the variability of the full sequence, a match cutoff
of 98% similarity with 95% coverage was used as the identification criteria.
Those sequences that did not match a human oral taxon sequence were subject
to BLASTN analysis against all sequences at the Ribosomal Database Project
(RDP; release 10, update 3) (11) and Greengenes (16). Because many clone
queries can match the same RDP or Greengenes subject match, a set of unique
reference matches was generated. Because this set could contain multiple entries
representing a single phylotype, the external database match sequences were
clustered as described below. Unique human oral taxon numbers were assigned
to each unique phylotype. The clones that did not meet the match criteria to the
HOMD, RDP, or Greengenes were clustered into novel taxa defined by the 98%
identity with 95% coverage criteria. Clustering was performed by first sorting the
clones by length. The first clone was considered a novel taxon and placed in a first
taxon folder, and its sequence was declared a reference sequence. Each succeed-

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TABLE 1. Phylogenetic distribution of 619 taxa in HOMD version 10

No. (%) of:
Phylum

Named speciesb

Unnamed
cultivated taxac

Unnamed
uncultivated taxad

Bacteria
Firmicutes
Bacteroidetes
Proteobacteria
Actinobacteria
Spirochaetes
Fusobacteria
TM7
Synergistetes
Chlamydiae
Chloroflexi
SR1

227 (36.7)
107 (17.3)
106 (17.1)
72 (11.6)
49 (7.9)
32 (5.2)
12 (1.9)
10 (1.6)
1 (0.2)
1 (0.2)
1 (0.2)

120 (52.9)
39 (36.4)
70 (66.0)
37 (51.4)
11 (22.4)
12 (37.5)
0 (0.0)
2 (20.0)
1 (100.0)
0 (0.0)
0 (0.0)

45 (19.8)
27 (25.2)
9 (8.5)
25 (34.7)
3 (6.1)
4 (12.5)
0 (0.0)
0 (0.0)
0 (0.0)
0 (0.0)
0 (0.0)

62 (27.3)
41 (38.3)
27 (25.5)
10 (13.9)
35 (71.4)
16 (50.0)
12 (100.0)
8 (80.0)
0 (0.0)
1 (100.0)
1 (100.0)

Archaea
Euryarchaeota

1 (0.2)

0 (0.0)

0 (0.0)

113 (18.3)

213 (34.4)

Total

619 (100)

1 (100.0)
293 (47.3)

Taxa refer to named species and to phylotypes with or without cultivable members. Phylotypes are defined as clusters whose members have 98.5% full 16S rRNA
sequence similarity. The data in this table are based on those in HOMD version 10.
b
Named species are those with validly published names.
c
Unnamed cultivable taxa are phylotypes that have at least one extant isolate.
d
Uncultivated taxa are phylotypes known only from clone sequences.

of the taxon observed in this study, and a symbol indicating

each taxons naming and cultivation status.
Firmicutes and Tenericutes. The phylum Tenericutes and the
classes Bacilli and Erysipelotrichia of the phylum Firmicutes are
shown in Fig. 1. The taxa belonging to the Firmicutes class
Clostridia are shown in Fig. 2 and 3.
The class Bacilli (Fig. 1, clade designated with an encircled
1) contains 86 taxa. It includes the genus Streptococcus,
whose members are the most abundant bacterial species in the
mouth. The related, but less frequently studied, genera
Abiotrophia, Gemella, and Granulicatella are also extremely
common, and three species from these genera were among the
10 taxa most frequently detected in our clone libraries. Abiotrophia and Granulicatella require the addition of pyridoxal to
grow on blood agar media and were formerly known as the
nutritionally variant streptococci (12, 55).
The class Erysipelotrichia (Fig. 1, clade designated with an
encircled 3) contains the following four oral organisms: Bulleidia extructa, Solobacterium moorei, Erysipelothrix tonsillarum,
and Lactobacillus [XVII] catenaformis. The meaning of the
roman number in square brackets is explained below.
The Firmicutes class Clostridia is shown in Fig. 2 and 3. For
the taxonomy of the Clostridia, a widely used classification is
that described by Collins et al. (13). Therefore, in the Clostridia
trees, Eubacterium infirmum is written Eubacterium [XI][G-1]
infirmum to indicate that it is a misnamed Eubacterium sp.
falling in Collins cluster XI and that it and neighboring taxa
represent a novel genus, as yet unnamed, designated [G-1].
NCBI, and RDP taxonomies, for the most part, remain based
on historic names rather than phylogenetic position, even when
genera are known to be polyphyletic, thus creating illogical
placements at the family and higher taxonomic levels. The
major oral clades, corresponding roughly to family-level divisions, are marked in Fig. 2 as follows: Collins cluster XI (en-

circled 1); the family Peptostreptococcaceae (encircled 2);

Collins cluster XIII (encircled 3); a novel family level cluster,
with no named species, designated F-1 (encircled 4); Collins
cluster XV, the Eubacteriaceae sensu stricto (encircled 5);
Collins cluster XIVa, the Lachnospiraceae (encircled 6); the
Peptococcaceae (encircled 7); Collins cluster VIII, the Syntrophomonadaceae (encircled 8); and a novel family-level
cluster, with no named species, designated F-2 (encircled 9).
The largest family of the Clostridia is the Veillonellaceae (previously called Acidaminococcaceae), as shown in Fig. 3. The
vast majority of human oral Clostridia fall in the families Lachnospiraceae, Peptostreptococcaceae, and Veillonellaceae.
All members of the Veillonellaceae, shown in Fig. 3, are
Gram negative and include the genera Anaeroglobus, Centipeda, Dialister, Megasphaera, Selenomonas, and Veillonella. It is
striking that a clade of Gram-negative organisms occurs in an
otherwise Gram-positive phylum.
Tenericutes. The phylum Tenericutes (Fig. 1, designated with
an encircled 4) has recently been created and was previously
the class Mollicutes within the Firmicutes (38). Mycoplasma
species have been detected in the saliva of 97% of individuals
(68). Relatively few mycoplasmas have been detected in the
clone libraries reported here, but representatives of Mycoplasma hominis, Mycoplasma salivarium, and Mycoplasma faucium were found. Tenericutes [G-1] sp. oral taxon 504 is very
deeply branching and has tentatively been placed with the
Tenericutes awaiting further information.
Actinobacteria. The phyla Actinobacteria and Fusobacteria
are shown in Fig. 4, and their clades are marked by an encircled 1 and an encircled 5, respectively. Out of eight named
Actinobacteria orders, oral taxa have thus far been found only
in the orders Actinomycetales (Fig. 4, encircled 2), Bifidobacteriales (encircled 3), and Coriobacteriales (encircled 4).
Actinomycetales (Fig. 4, encircled 2) include the genera

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FIG. 1. Neighbor-joining tree for human oral taxa in the phylum Tenericutes and the classes Bacilli and Erysipelotrichia of the phylum Firmicutes.
The name of each taxon is followed by the oral taxon number, clone or strain number, GenBank accession number, the number of clones out of

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Burkholderia, Ralstonia, Delftia, Variovorax, and Leptothrix.

Most of these genera are aerobes. Gammaproteobacteria include oral taxa in the following five families: Xanthomonadaceae (Fig. 6, encircled 1), Cardiobacteriaceae (encircled
2), Pseudomonadaceae (encircled 3), Moraxellaceae (encircled 4), Enterobacteriaceae (encircled 5), and Pasteurellaceae (encircled 6). The Xanthomonadaceae family (Fig. 6,
encircled 1) includes Stenotrophomonas maltophilia, which is
an ubiquitous environmental organism that is an opportunistic
pathogen which can cause nosocomial infections (57) but also
can contaminate laboratory reagents and show up in cloning
libraries as an artifact, as can many members of the Proteobacteria (63). BLAST interrogation of the GenBank database with
Haemophilus parainfluenzae sequences frequently results in a
match with accession no. AB098612, labeled Terrahaemophilus aromaticivorans, an isolate from petroleum sludge. This
species has not been formally proposed and, in our opinion,
was likely a human oral H. parainfluenzae contaminant in their
sample.
Deltaproteobacteria include species in the genera Desulfovibrio, Desulfomicrobium, Desulfobulbus, and Bdellovibrio. Epsilonproteobacteria include the genera Campylobacter, Helicobacter, and the misnamed Bacteroides ureolyticus, which falls
just inside or adjacent to the genus Campylobacter (66).
The Spirochaetes phylum is shown as the clade marked with
an encircled 1 in Fig. 7. All human oral taxa identified to
date in the phylum Spirochaetes are members of the genus
Treponema. Over 70% of the 49 identified taxa have thus far
resisted cultivation. The number of proposed taxa has decreased from 57 to 49 relative to our initial description of the
oral treponemes (18), as some of the taxa with greater than
98.5% 16S rRNA sequence similarity were combined.
The Chlamydia phylum is marked with an encircled 2 in
Fig. 7. Chlamydophila pneumoniae has been detected in dental
plaque (39a) and is a recognized lung pathogen. Chlamydia
were not observed in our cloning studies, perhaps because the
common 9 to 27 (E. coli numbering) 16S rRNA PCR forward
primer contains two mismatches with Chlamydia sequences
(24). Use of the YM3 forward primer set (24) may remedy
this problem in future studies.
The Chloroflexi phylum is marked with an encircled 3 in
Fig. 7. A single Chloroflexi phylotype has been identified. Chloroflexi are abundant in studies of wastewater treatment sludge
(8). A closely related phylotype (95% similarity) has been
found in the canine oral cavity (F. E. Dewhirst, unpublished
observation), suggesting there are multiple mammalian hostassociated species in the phylum Chloroflexi.
Synergistetes taxa are shown in the clade marked with an
encircled 4 in Fig. 7. Ten taxa in the phylum Synergistetes
have been identified (34, 35). This recently described phylum
includes a number of genera and phylotypes that have been

34,879 that were identified as this taxon, and a symbol indicating that the taxon is a named species (F), an unnamed cultivated taxa (f), or an
uncultivated phylotype (). The tree was constructed with MEGA 4.3 using the Jukes and Cantor correction neighbor-joining distance matrix.
Comparisons with missing data were eliminated pairwise. The numbers to the left of the branches indicate the percentage of times the clade was
recovered out of 1,000 bootstrap resamplings. Only bootstrap percentages greater than 50 are shown. Roman numerals in square brackets following
a genus name indicate Collins Clostridia cluster numbers (17). Major clades are marked as follows: encircled 1, Bacilli; encircled 2, Firmicutes;
encircled 3, Erysipelotrichia; and encircled 4, Tenericutes (previously know as the class Mollicutes within Firmicutes).

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Actinomyces, Rothia, Kocuria, Arsenicicoccus, Microbacterium,

Propionibacterium, Mycobacterium, Dietzia, Turicella, and
Corynebacterium. Bifidobacteriales (Fig. 4, encircled 3) include the genera Bifidobacterium, Gardnerella, Scardovia, and
Parascardovia. These genera are found primarily in dental caries and in denture plaque (40), except for Gardnerella, which is
isolated primarily from the vagina. Coriobacteriales (Fig. 4,
encircled 4) include the following genera with oral members:
Atopobium, Cryptobacterium, Eggerthella, Olsenella, and Slackia
(17).
Fusobacteria. The phylum Fusobacteria includes the following two genera frequently detected in the mouth: Fusobacterium and Leptotrichia. An unnamed and uncultivated genus,
Fusobacteria [G-1], contains two taxa, with Fusobacteria [G-1]
sp. oral taxon 220 being relative common with 47 clones detected.
Bacteroidetes. The 107 Bacteroidetes taxa, shown in Fig. 5,
fall into the genera Prevotella, Bacteroides, Porphyromonas,
Tannerella, Bergeyella, Capnocytophaga, and eight that are unnamed.
Prevotella is the largest genus, with approximately 50 species.
The majority have been cultivated. The clade marked with an
encircled 0 in Fig. 5, including Prevotella tannerae through
Prevotella sp. oral taxon 308, warrants separate genus status, as
it shares less than 80% 16S rRNA similarity with the main
cluster of Prevotella (oral taxa 313 through 304).
In Fig. 5, eight currently unnamed genera (comprised of 1 to
5 phylotypes each) are marked by encircled 1 through encircled 8. These taxa are deeply branching and have no closely
related named species. Bacteroidaceae [G-1] sp. oral taxon 272
(encircled 1) is both cultivable and common, with 210 clones
observed. Three strains in the Moores collection labeled Prevotella zoogleoformans have been identified as belonging to oral
taxon 272. Bacteroidales [G-2] sp. oral taxon 274 (encircled
2) is also cultivable and common, with 51 clones observed
and an isolate designated Bacteroides D59 identified from
the Moores collection. The six remaining unnamed genera,
marked encircled 3 through encircled 8, have, as yet, no
cultivated isolates, but Bacteroidetes [G-3] sp. oral taxon 281 is
common with 54 clones in our collection.
Bergeyella zoohelcum, the only named species of the genus, is
a member of the canine oral microbiome and a human pathogen from dog bites (65). The two human oral Bergeyella taxa
are uncultivated.
Proteobacteria. All five classes of Proteobacteria, Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Deltaproteobacteria, and Epsilonproteobacteria, contain taxa detected
in the human oral cavity. These taxa are shown in Fig. 6, where
the classes are labeled with their corresponding Greek letters.
Betaproteobacteria include the genera Neisseria, Eikenella,
Kingella, Simonsiella, Achromobacter, Bordetella, Lautropia,

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FIG. 2. Neighbor-joining tree for human oral taxa in the class Clostridia of the phylum Firmicutes. Labeling and methods used are as described
in Fig. 1. Major clades are marked as follows: encircled 1, Collins XI; encircled 2, Peptostreptococcaceae; encircled 3, Collins XIII; encircled
4, unnamed family F1; encircled 5, Collins XV, Eubacteriaceae; encircled 6, Collins XIVa, Lachnospiraceae; encircled 7, Peptococcaceae;
encircled 8, Collins XIII, Syntrophomonadaceae; and encircled 9, unnamed family F2.

previously misclassified as belonging to either the Deferribacteres or Firmicutes (28). Oral strains and clones fall into two
main groups, with one which is readily cultivable and includes
the named species Jonquetella anthropi (34) and Pyramidobacter piscolens (19). Most oral sequences fall into the
second group (oral taxon 363 through 359), which until recently had no cultivable representatives. However, Vartoukian
et al. (67) have successfully cultured a member of this group by

a combination of coculture and colony hybridization-directed

enrichment. Members of this phylum are selectively amplified
and appear in significant numbers in 16S rRNA gene libraries
generated with Spirochaeta/Synergistetes selective primer pair
F24/M98 (see Table S2 in the supplemental material).
The TM7 phylum is marked with an encircled 5 in Fig. 7.
There are 12 TM7 phylotypes of this phylum shown in Fig. 7.
The name TM7 comes from Torf, mittlere Schicht, clone 7 (or

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HUMAN ORAL MICROBIOME

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peat, middle layer, clone 7), a study of organisms in a German

peat bog (53). Organisms in this phylum are frequently detected in many environments (29), but despite the efforts of
several laboratories, no members of this phylum have been
cultivated, except as microcolonies (23).
The SR1 phylum is marked with an encircled 6 in Fig. 7.
The Sulfur River 1 lineage is now recognized as a phylum
distinct from OP11 (Obsidian Pool, candidate division 11),
with which it was previously grouped (27). Earlier publications
referred to SR1 sp. oral taxon 345 as clone X112 in phylum
OP11 (47). Clones of oral taxon 345 have been obtained from
several distinct individuals. This species is enriched in 16S
rRNA gene libraries generated with Bacteroidetes/TM7/SR1
selective primer pair F24/F01 (see Table S1 in the supplemental material). A closely related species (95% similarity) has
been found in the oral cavities of cats and dogs (F. E. Dewhirst,
unpublished observation), indicating that there are multiple
mammalian host-associated species in the SR1 phylum.
Web-based Human Oral Microbiome Database. The taxonomic scheme and sequence database described above formed
the basis for creating a publically accessible web-based Human
Oral Microbiome Database (http://www.homd.org). It is a resource for phylogenetic, taxonomic, genomic, phenotypic and
bibliographic data related to the human oral microbiome and
is supported by a contract from the National Institute for
Dental and Craniofacial Research to facilitate research by the

oral microbiome community. The database was formally

launched on 1 March 2008. The HOMD hardware, program
architecture, and website navigation have been described in a
separate publication (9). Briefly, the HOMD contains a taxon
description page for each taxon with full taxonomy, its status as
a named species, an unnamed isolate or an uncultivated phylotype, its type or reference strain number, and links to the
literature. As a taxon moves from a phylotype, to an isolate,
and to a named species, the database tracks the name and
status of the bacterium and provides a stable link to the literature. The community can provide input to taxon descriptions
and statuses using the HOMD interface. The HOMD contains
a BLASTN tool for identifying the 16S rRNA sequences of
isolates or clones. Hundreds of sequences can be submitted at
one time, and the results can be downloaded as an Excel file
showing the top four hits in the database for each query sequence. For some groups of taxa, 16S rRNA gene sequence
analysis of partial sequences does not allow unequivocal identification at the species level. The HOMD BLASTN tool output allows easy detection of ambiguous identifications. It also
contains visualization and analysis tools for examining the partial and completed genomes for any taxa of the human oral
microbiome. It is likely that genomes for over 300 oral taxa will
be available on the HOMD by the end of the Human Microbiome Project (51).

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FIG. 3. Neighbor-joining tree for human oral taxa in the Veillonellaceae (previously Acidaminococcaceae) family of the class Clostridia of the
phylum Firmicutes. Labeling and methods used are as described in Fig. 1.

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FIG. 4. Neighbor-joining tree for human oral taxa in the phyla Actinobacteria and Fusobacteria. Labeling and methods used are as described
in Fig. 1. Major clades are marked as follows: encircled 1, phylum Actinobacteria; encircled 2, order Actinomycetales; encircled 3, order
Bifidobacteriales; encircled 4, order Coriobacteriales; and encircled 5, phylum Fusobacteria.
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FIG. 5. Neighbor-joining tree for human oral taxa in the phylum Bacteroidetes. Labeling and methods used are as described in Fig. 1. Encircled
0 through 8 symbols refer to clades discussed in the text.
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FIG. 6. Neighbor-joining tree for human oral taxa in the phylum Proteobacteria. Labeling and methods used are as described in Fig. 1. The
Proteobacteria classes are marked with the corresponding Greek letters.
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Clone analysis. To validate and expand the species included

in the HOMD and to better understand the diversity and taxon
distribution of organisms in the human oral microbiome,
36,043 clones from 633 oral 16S rRNA gene libraries constructed and sequenced in our laboratories were analyzed.
Following vector removal, screening for lengths of 300 bases,
and chimera checking using multiple programs, 1,290 sequences were rejected, leaving 34,753 for analysis. The clones
identified as potentially chimeric by the program Chimera
Slayer are listed in Table S3 in the supplemental material. A
total of 125 were validated as chimeras. Those not validated
include intragenus, intraspecies potential chimeras. The ma-

jority of these nonvalidated chimeras were found multiple

times, which also suggests that they are not chimeras. BLASTN
analyses of the remaining clones against the HOMD Reference Sequence Set version 10 yielded identification of 89.1% of
the clones in 525 oral taxa. Those not identified in the HOMD
were analyzed by BLASTN comparison to the more than
70,000 16S rRNA sequences in RDP (11) and 302,066 sequences in the Greengenes prokMSA unaligned set (16). Using the RDP and Greengenes databases, an additional 8.7% of
clones were matched to nonself sequences. The 2.2% of the
clones which remained unidentified were clustered into 325
novel taxa, 220 of which were singletons. The clone analysis

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FIG. 7. Neighbor-joining tree for human oral taxa in the phyla Spirochaetes, Chlamydiae, Chloroflexi, Synergistetes, TM7, and SR1. Labeling and
methods used are as described in Fig. 1. The phyla are labeled as follows: encircled 1, Spirochaetes; encircled 2, Chlamydiae; encircled 3,
Chloroflexi; encircled 4, Synergistetes; encircled 5, TM7; and encircled 6, SR1.

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J. BACTERIOL.
TABLE 2. Phylogenetic distribution of 34,753 oral clones
% of taxa that represent:

Phylum

Total

% of clones that represent:

Named
species

Unnamed
isolates

Uncultivated
phylotypes

No. of clones (%)

494 (41.9)
237 (20.1)
153 (13.0)
133 (11.3)
73 (6.2)
42 (3.6)
18 (1.5)
15 (1.2)
4 (0.3)
3 (0.2)
1 (0.1)
1 (0.1)
1 (0.1)
1 (0.1)
4 (0.3)

20.6
33.3
19.0
35.3
9.6
23.8
11.1
0.0
75.0
0.0
0.0
0.0
0.0
0.0
100.0

8.7
3.0
16.3
14.3
2.7
7.1
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0

70.6
63.7
64.7
50.4
87.7
69.0
88.9
100.0
25.0
100.0
100.0
100.0
100.0
100.0
0.0

23,354 (67.212)
3,909 (11.250)
2,571 (7.403)
2,641 (7.572)
577 (1.671)
1,356 (3.893)
209 (0.605)
85 (0.244)
10 (0.028)
13 (0.037)
7 (0.020)
1 (0.003)
1 (0.003)
1 (0.003)
19 (0.054)

78.7
72.7
58.6
55.7
33.8
76.6
12.4
0.0
90.0
0.0
0.0
0.0
0.0
0.0
100.0

7.3
3.8
24.1
20.3
4.3
4.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0

14.1
23.6
17.3
24.0
61.9
19.4
87.6
100.0
10.0
100.0
100.0
100.0
100.0
100.0
0.00

1,179 (100)

24.0

8.4

67.7

34,753 (100.000)

73.3

8.8

17.8

yielded a total of 654 additional taxa not included in the

HOMD version 10. Because novel taxa based on singletons are
suspect compared to those seen multiple times, a more conservative figure excluding the 220 singletons is 434 novel taxa.
These additional taxa will be added to the HOMD when they
meet the strict criteria described below.
A total of 94 of 619 taxa in the HOMD version 10 were not
represented by any clone. The list of taxa not found is provided
in Table S5 in the supplemental material. This group included
most of the extrinsic pathogens we had added to the HOMD
for completeness, such as Bordetella pertussis, Corynebacterium
diphtheriae, Mycobacterium tuberculosis, and Neisseria gonorrhoeae. Other taxa not seen included those which would not
have been expected to have been amplified with the universal primers used, such as Methanobrevibacter oralis (an archaeon) and Chlamydophila pneumoniae.
The phylogenetic distribution of clones in various phyla or
divisions is shown in Table 2. The clones were found in 11 of
13 phyla included in the HOMD version 10. As the primers
used do not amplify Chlamydiae or Archaea, the absence of
clones representing these taxa was expected. Clones for the
phyla Deinococcus (3 clones), Acidobacteria (1), and Cyanobacteria (1) were found. The clones for these phyla may represent
transient exogenous bacteria. Nineteen clones representing
four plants (chloroplast) were detected. As virtually all humans
eat plants, it is not surprising that plant chloroplast 16S rRNA
sequences should be detected in the oral cavity. Species identified included Triticum aestivum (wheat) and Manihot esculenta (cassava, source of tapioca). For each phylum, the percentage of taxa and clones that represent named species,
unnamed taxa with cultivable isolates, and uncultivated phylotypes is presented in Table 2. When analyzed by clone distribution, 82% fall into cultivable named and unnamed taxa.
When analyzed by taxa distribution, the percent cultivated
drops to 32% (40% if singleton clone taxa are removed). This
divergence in percent cultivated by clone or taxa count indicates that we have had great success culturing the most common species but have not yet identified isolates for the rarer

Named
species

Unnamed
isolates

Uncultivated
Phylotypes

taxa. The microbiologic community has cultured between 29%

and 50% of the oral Firmicutes, Proteobacteria, Bacteroidetes,
Actinobacteria, and Fusobacteria taxa. Less than 12% of taxa in
the Spirochaetes and Synergistetes, however, have been cultured. TM7, SR1, and Chloroflexi have, as yet, no cultivated
members (the phylum Chloroflexi has cultivated members but
no cultivated human oral species). The rarer taxa, such as SR1,
are present in the clone pool at level of only 1/5,000. This
implies that even if the SR1 taxon is only moderately difficult
to cultivate, it will still be challenging to find an isolate because
of its rarity.
A plot of the relative abundance of clones in each of the
1,179 oral taxa is shown in Fig. 8. Veillonella parvula was the
first ranked taxon, with 2,304 clones or 6.6% of the total clones
observed. The complete listing of the rank abundance and
relative abundance of each taxon is presented in Table S6 in
the supplemental material. Because the 34,753 clones are from
studies of different disease and health states, different oral
sites, and different primer pairs, the data set cannot be used to
infer the underlying population structure of the oral cavity.
We therefore do not attempt to estimate the number of species

FIG. 8. Rank abundance graph for 34,753 16S rRNA clones obtained from oral samples in 1,179 taxa. Clones were placed in taxa on
the basis of 98% BLASTN identities. The first ranked taxon was
Veillonella parvula, with 2,304 clones. Ranks 769 to 1,179 were singletons.

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Firmicutes
Proteobacteria
Bacteroidetes
Actinobacteria
Spirochaetes
Fusobacteria
Synergistetes
TM7
Tenericutes
Deinococcus
SR1
Chloroflexi
Acidobacteria
Cyanobacteria
Plant chloroplast

No. of taxa
(%)

VOL. 192, 2010

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greater than 98.5% similar to that of the type strain of the

species. (vi) If there are multiple sequences in GenBank for a
species, the longest and most accurate must be identified to
represent the species. We expect that the majority of the taxa
identified in the clone analysis will be validated and added in
updates to the HOMD by January 2011.
The human oral microbiome has been extensively studied, is
being examined as part of the Human Microbiome Project, and
will continue to be examined in the future using ever more
powerful sequencing technologies. We have created the Human Oral Microbiome Database taxonomic framework, with
oral taxon numbers to facilitate communication between investigators exploring the diversity of the oral microbiome, as
seen by 16S rRNA gene-based methods. It is critical that investigators can point to curated stably designated taxa rather
than taxonomically undefined clone sequences in disseminating research findings. Investigators with oral strain or clone 16S
RNA sequences can now definitively identify the vast majority
of them by BLASTN analysis against the reference sets available at the HOMD website (www.homd.org). Analysis of approximately 35,000 clone sequences has allowed validation of
the initial 619 species in the HOMD and identified more than
434 additional named and unnamed taxa to be added to the
HOMD once full 16S rRNA sequences are obtained and the
other criteria discussed above are met. The HOMD is the first
example of a curated human body site-specific microbiome
resource. Predominant members of the oral microbiome can
also be found in fewer numbers at other body sites, such as the
skin, gut, and vagina, and thus, the HOMD can be useful to the
entire Human Microbiome Project and infectious disease communities. One can foresee the development of additional body
site-specific curated microbiome resources based on the
HOMD model or the framework of the HOMD to be expanded to include the entire human microbiome.
ACKNOWLEDGMENTS
This work has been supported in part by contract U01 DE016937
and grants DE015847 and DE017106 from the National Institute of
Dental and Craniofacial Research and a supplement to grant
DE016937 from the American Recovery and Reinvestment Act of
2009.
We thank the following investigators for providing clinical samples
or DNA for the construction of clone libraries: Ashraf F. Fouad, Ann
L. Griffen, Anne D. Haffajee, Onir Leshem, Eugene J. Leys, Harlan J.
Shiau, Sigmund S. Socransky, and Thomas R. Flynn. We thank Julia
Downes for helpful discussions. We thank all of our colleagues who
have deposited 16S rRNA gene sequences for oral strains in public
databases.
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in the oral cavity with these data. Furthermore, the question of

how many species are present in the human oral cavity? is
tricky, because the oral cavity is an open system where exogenous microorganisms from the environment are continually
introduced by eating, drinking, and breathing. One answer to
the question of how many microbial species would be found by
exhaustive sampling of the oral cavities of the human population over time is all microbial species present in the earths
biosphere. A more straightforward question which can be answered for this data set is, How many taxa are needed to
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sampled? The answers are 259, 413, 655, and 875 taxa, respectively.
Transient versus endogenous species. Because there is a
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flora (54). Unfortunately, the distinction between transient
species and endogenous species cannot be deduced directly
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comparing the human studies with environmental studies to
determine the frequency with which clones of a particular
genus are recovered as host associated or as environment associated. For example, species in the genus Prevotella have
been found only in the microbiota of mammals, whereas species in the genus Sphingomonas are generally free-living species found in the environment in cloning studies of lake sediments, soils, etc. Table S7 in the supplemental material
presents the categorization of the 169 genera currently included in the HOMD as strongly host associated, weakly host
associated, or environmental based on a qualitative analysis of
clone sequence sources. The degree of host association for
various genera differs widely by phylogenetic position, as essentially all genera from the phylum Bacteroidetes are host
associated, while nearly all genera from the Alphaproteobacteria are thought to be environmental transients.
Extended Reference Set for BLAST analyses. Reference sequences for the 654 additional taxa identified in the clone
analysis have been added to those for the 619 HOMD taxa to
generate the Extended Reference Set (version 1), which is
available for download or use in BLAST analysis at the
HOMD website. Many sequences from the clone analysis are
only 500 bases long, and thus, the Extended Reference Set,
unlike the HOMD Reference Set 10, is composed of both full
and partial sequences. The Extended Reference Set is useful
for BLAST analysis of clone and other 5 500-base sequences
but must be used with caution for analysis of full-length sequences.
Addition of taxa to the Human Oral Microbiome Database.
The 434 additional taxa identified in the clone analysis (excluding singletons) described above are candidates for addition
to the HOMD. To be added, however, each taxon must meet
the following criteria. (i) It must have been seen more than
once, hence the exclusion of novel singletons. (ii) A near fulllength (1450-base) 16S rRNA gene sequence must be available for the taxon. (iii) The full sequence must be less than
98.5% similar to previously defined HOMD taxa. (iv) The full
sequence must pass chimera and sequence quality screens. (v)
If the sequence is for a named species, the sequence must

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